Biolistic transformation of the human pathogenic fungus Coccidioides immitis

Abstract

Coccidioides immitis is a fungal respiratory pathogen of humans. The infectious, saprobic phase of the microbe resides in soil of southwestern desert regions of the United States. The genetics of this pathogen are essentially undeveloped. Progress in studies of the molecular biology of C. immitis have been hampered by the absence of a transformation system. We report here the first stable integration of plasmid DNA into chromosomes of C. immitis by the method of biolistic DNA delivery. A previously-described pAN7-1 plasmid and a newly-constructed C. immitis URA5 gene disruptant (pU5H-1) were used to transform C. immitis strain 735. The 6.8-kilobase (kb) pAN7-1 plasmid and the 10.1-kb pU5H-1 construct both contained the hygromycin phosphotransferase gene ( hph) which encodes a protein that inactivates hygromycin B (HmB) by phosphorylation. Germinated, saprobic phase spores (arthroconidia) and parasitic phase cells (spherules) of C. immitis were subjected to bombardment by gold particles coated with either the linearized pAN7-1 or the circular pU5H-1 plasmid DNA (50 μg DNA per 25 mg gold particles). A single pAN7-1 and two pU5H-1 transformants were initially selected from approximately 10 7 cells on the basis of their ability to grow on glucose–yeast extract agar supplemented with 60–80 μg/ml HmB. The transformants showed resistance to HmB up to a concentration of approximately 320 μg/ml of media, compared to the parental strain which was unable to grow in the presence of 40 μg HmB/ml of media. Southern hybridization analyses of the transformants suggested that both the pAN7-1 and pU5H-1 DNAs were integrated into chromosomal DNA of C. immitis as tandem repeats. After animal passage, all transformants showed mitotic stability of the integrated DNA. This newly-developed transformation system permits genetic manipulations of C. immitis which were not previously possible.